Project #2: OXIDANT STRESS AND ASCORBIC ACID PROCESSING IN DIABETES The objective of the Program Project is to investigate the role of oxidative and carbonyl stress in the pathogenesis of diabetic complications. In particular, this project (Project 2) seeks to test the hypothesis that the oxidative stress resulting from high glucose in tissue culture and the diabetic rat is, in contrast to the diabetic human, primarily transition metal and aldose reductase dependent. Furthermore, we hypothesize many of the drugs thought to act as aldose reductase inhibitors, anti-glycating agents, growth factors, NO synthase, PKC and other inhibitors have beneficial effects in the rat as transition metal chelators and/or antioxidants. We propose to test these hypotheses by using a very powerful probe for assessment of oxidative stress in vivo, i.e 6-deoxy-6- fluoro ascorbic acid (F-ascorbate) in conjunction with 750 MHz 19F- NMR-spectroscopy, while at the same time providing the first biochemical insight into ascorbate catabolism in vivo and its modulation by diabetes. 1. To determine the chemical nature and mechanism of formation of F- ascorbate degradation products formed in vitro in low and high glucose environment. 2: To determine how the metabolic pathways of F-ascorbate degradation in cells implicated in diabetic complications are influenced by high glucose environment, and 3. To determine how diabetes in the rat and the human affects ascorbate processing, and whether uncovered abnormalities predict development of diabetic complications in the rat and DCCT patient. Using this novel approach, we expect to clarify the critical question: Is the reason why a large number of drugs have been successful in the diabetic rat but have failed in the human due to normalization of oxidative stress that is specific to the diabetic rat and of such overwhelming intensity that it masks other mechanisms of diabetes induced complications in the human. If confirmed, this outcome would have a profound effect on the future development of anti-diabetic therapies based on the rat model of hyperglycemia.